The goal of this proposal is to describe the mechanism of anaphase spindle elongation (anaphase B) at a structural, and molecular level using as functional model systems the highly ordered, spindles isolated from diatoms and spindles in permeabilized yeast cells. Spindles elongate in vitro because microtubules of one half spindle slide over the microtubules of the other half spindle. In this proposal we want to understand the organization and composition of the zone of microtubule overlap, which is the site of force transduction during spindle elongation. We will utilize a new isolated functional spindle that can be prepared in bulk to make antibodies against spindle midzone (zone of microtubule overlap) components. We are interested in preparing antisera against proteins that are involved in the mechanochemistry of spindle elongation. We have developed a series of strategies to identify and enrich for these proteins using selective extractions, cross-linking to tubulin, an antibody that inhibits function, or the detection of of spindle protein phosphorylations to identify classes of proteins that are important for the mechanism of anaphase B. These antibodies will be used as functional blockers of our in vitro cell model, to localize components in the spindle, as aids in their purification, and to clone the relevant genes from diatom and yeast Lambda gt11 expression libraries.. We have found an antibody (the LAGSE antibody which recognizes an epitope in the kinesin head domain) that blocks spindle elongation in vitro and will use it to identify the anaphase B motor. To understand the organization of the spindle midzone at an ultrastructural level, we propose two kinds of analysis: 1) studies of native spindle structure using isolated spindles fixed by rapid freezing, and 2)immunocytochemical localization of overlap zone components after various physiological treatments. 'ne anaphase B motor and many other components of the zone of microtubule overlap should be found in all eukaryotic cells. We propose to use the antibodies we generate against diatom spindle associated proteins to screen yeast mitotic extracts and yeast expression libraries to determine whether these same proteins are also found in yeast. Given the wealth of new mutations that affect mitosis in budding yeast, we will develop an in vitro cell model that can be used to analyze spindle elongation in Saccharomyces cerevisiae. We will also determine whether the LAGSE antibody will recognize kinesin-like proteins and inhibit spindle elongation in vitro in permeabilized Schizosaccharomyces pombe cells. The regulation of mitosis is a topic of major medical interest since uncontrolled cell division is at the heart of the cancer problem and inaccurate chromosomal segregation (aneuploidy) is causal in several congenital malformations and a major cause of premature termination of pregnancy. An improved understanding of mitosis should eventually lead to new approaches to cancer chemotherapy and to control of abnormal chromosome segregation.